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Volume 110 (1991)
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- Issue 2 Pages 163-
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Volume 110, Issue 2
Displaying 1-27 of 27 articles from this issue
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Shuntaro Hara, Ichiro Kudo, Keizo Inoue1991 Volume 110 Issue 2 Pages 163-165
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat group II phospholipase A2 added exogenously to A23187-activated HL-60 granulocytes augmented their production of prostaglandin E2. Human group II phospholipase A2 and porcine group I phospholipase A2 augmented the prostaglandin E2 production in a similar manner. No significant increase in prostaglandin E2 production was observed when cells were treated with purified phospholipase A2 in the absence of A23187. Extracellular phospholipase A2 at inflamed sites may contribute to the generation of pro-inflammatory lipid mediators by hydrolyzing the cellular phospholipids of activated inflammatory cells.View full abstractDownload PDF (304K) -
Takanori Kigawa, Shigeyuki Yokoyama1991 Volume 110 Issue 2 Pages 166-168
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA continuous cell-free protein synthesis system [Spirin et al. (1988) Science 242, 1162-1164] was modified so as to be suitable for coupled transcription-translation, a process useful for obtaining products of cloned genes or cDNAs. A reaction chamber equipped with an ultrafiltration membrane was newly designed and an HPLC pump was used to supply a low molecular weight substrate solution at a constant rate to the viscous reaction mixture in the chamber. By using an Escherichia coli S30 extract in this modified flow system (1 ml), coupled transcription-translation could be continuously performed for 17 h, the synthesized chloramphenicol acetyltransferase (_??_ 0.1 mg) being subsequently eluted through the chamber's membrane and then purified.View full abstractDownload PDF (2673K) -
Haruo Suzuki, Hirokazu Koyama, Yasuzo Nishina, Kyosuke Sato, Kiyoshi S ...1991 Volume 110 Issue 2 Pages 169-172
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSResonance Raman (RR) spectra of purple intermediates of L-phenylalanine oxidase (PAO) with non-labeled and isotopically labeled phenylalanines as substrates, i. e., [1-13C], [2-13C], [ring-U-13C6], and [15N]phenylalanines, were measured with excitation at 632.8 nm within the broad absorption band around 540 nm. The spectra obtained resemble those of purple intermediates of u-amino acid oxidase (DAO). The isotope effects on the 1, 665cm-1 band with [15N] or [2-13C]phenylalanine indicate that the band is due to the C=N stretching mode of an imino acid derived from phenylalanine, i. e., α-imino-β-phenylpropionate. The intense band at 1, 389cm-1 is contributed to by the CO2- symmetric stretching and C-CO2- stretching modes of α-imino-β-phenylpropionate. The 1, 602cm-1 band, which does not shift upon isotopic substitution of phenylalanine, corresponds to the 1, 605cm-1 band of DAO purple intermediates and was assigned to a vibrational mode associated with the C(10α)=C(4α)-C(4)=O moiety of reduced flavin. These results confirm that PAO purple intermediates consist of the reduced enzyme and an imino acid derived from a substrate, and suggest that the plane defined by C(10a)=C(4a)-C(4)=0 of reduced flavin and the plane containing H2N+=C-CO2- of an imino acid are arranged in close contact to each other, generating a charge-transfer interaction.View full abstractDownload PDF (413K) -
Tomoaki Tomiya, Yuzuru Sato, Yoshinori Murakami, Sumiko Nagoshi, Itsur ...1991 Volume 110 Issue 2 Pages 173-178
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHepatic ornithine decarboxylase (ODC) activity increases after partial hepatectomy and this activity is further stimulated by pharmacologic doses of glucagon and insulin. We now present data suggesting that glucagon and insulin stimulate ODC activity by distinct mechanisms. ODC activity increased progressively after partial hepatectomy and reached an initial peak at 4 h. Activity decreased to 50% of its peak value at 6 and 8 h and then rose progressively to a maximum at 12 h. Enzymatic activity was well correlated with the amount of hepatic immunoreactive ODC protein, thus suggesting that increased enzyme activity was due to increased amount of enzyme protein. Hepatic ODC mRNA increased gradually and continuously, reaching the maximal value by 12 h. In rats receiving glucagon after partial hepatectomy, ODC mRNA increased significantly by 2 h and enzyme immunoreactive protein and activity by 2 to 4 h as compared to controls. In contrast, insulin administration only induced a significant increase in enzyme immunoreactive protein and activity 10 to 12 h after partial hepatectomy. No significant changes in ODC mRNA level were observed. Our data suggest that the regulation mechanism of ODC induction following partial hepatectomy differs depending on the time after operation. Our data also suggest that while glucagon appears to regulate ODC activity by a transcriptional mechanism, insulin appears to operate at a post-transcriptional level.View full abstractDownload PDF (1687K) -
Hironobu Koga, Hidenori Yamada, Yukio Nishimura, Keitaro Kato, Taiji I ...1991 Volume 110 Issue 2 Pages 179-188
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDipeptidylcarboxypeptidase, endopeptidase, and carboxypeptidase activities of rat liver cathepsin B were investigated using soluble denatured protein substrates, reduced and S-(3-trimethylammonio)propylated proteins and their derivatives. It was found that the soluble denatured proteins were degraded mainly by the dipeptidylcarboxypeptidase activity and in a few cases by the endopeptidase and carboxypeptidase activities. The dipeptidylcarboxypeptidase activity showed broad substrate specificity with broad pH optimum at 4-6. A peptide having the α-carboxyl group amidated with methylamine could also be a good substrate for this activity. These results suggest that this activity is dependent not upon the dissociated α-carboxyl group at the P2' site but upon the hydrogen-bonding abilities of the α-imino moiety and the protonated or amidated α-carboxyl moiety at P2'. On the other hand, the endopeptidase and carboxypeptidase activities were observed in a few cases, suggesting that special amino acid sequences in the substrates are responsible for these activities. These activities showed sharp pH optima at 6 and seemed to prefer basic amino acid residues at P1 site. Therefore, we suppose that cathepsin B has a carboxyl group with a pKa of about 5.5 at the S1 subsite which more effectively interacts with a positive charge at the P1 site of the substrate at pH 6 than at pH 5. Based on these results, a model of the binding subsites of this enzyme is proposed.View full abstractDownload PDF (1102K) -
Fuminori Umenishi, Makoto Umeda, Kaoru Miyazaki1991 Volume 110 Issue 2 Pages 189-195
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMetalloproteinase inhibitors were surveyed with the culture media of 19 kinds of human tumor cell lines, using transin (rat stromelysin) as the target enzyme. This survey showed that most of the cell lines more or less secreted inhibitor activity, and that a human hepatoma cell line, HLE, secreted an extremely high inhibitor activity into the culture medium. Two kinds of metalloproteinase inhibitors were purified from the serum-free conditioned medium of HLE cells. The major inhibitor, which showed a single protein band with a molecular weight (Mr) of 21, 000 (21k) (nonreduced) or 24k (reduced) on SDS-polyacrylamide gel electrophoresis, was identified as TIMP-2 (tissue inhibitor of metallo-proteinases-2) by the analysis of its N-terminal amino acid sequence. The other was immunologically identified as TIMP. Purified TIMP-2 inhibited the activities of transin, matrin (pump-1), Mr 72k gelatinase, and interstitial collagenase with 1 : 1 stoichiometry. When the latent precursor form (Mr 57k) of transin was incubated with p-aminophenylmer-curie acetate as an activating reagent, TIMP-2 inhibited the conversion of the intermediate form (Mr 45k) into the mature enzyme (Mr 42k). This indicated that TIMP-2 regulates not only the activity of the mature enzyme but also the autolytic processing of the proenzyme. TIMP-2 also inhibited in vitro tumor invasion through reconstituted basement membrane (matrigel) in chemotaxis chambers, showing that the metalloproteinase inhibitors as well as the extracellular matrix metalloproteinases are involved in tumor invasion through basement membrane and other extracellular matrices.View full abstractDownload PDF (3118K) -
Nobumasa Yoshida, Kenzo Nakamura1991 Volume 110 Issue 2 Pages 196-201
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTuberous roots of the sweet potato are unusually rich in β-amylase, and the β-amylase polypeptides account for about 5% of the total soluble protein of the organ. Unlike p-amylases from other origins, the sweet potato β-amylase is a tetramer of identical subunits, and it also bears starch phosphorylase-inhibitor activity. A cDNA for the subunit of sweet potato β-amylase was obtained by immunological screening of an expression cDNA library constructed by the vector-primer and linker method using a plasmid vector containing tac-SP6 promoters. The SP6 transcript of a 2, 000 base-pair-long cDNA insert directed the synthesis in vitro of a precursor to the subunit of β-amylase which was identical in size with the mature subunit, and the β-amylase mRNA detected by Northern blot hybridization was identical in size with the SP6 transcript of the cDNA insert. The cDNA insert contained 1, 494 base pairs of an open reading frame which codes for the 499-amino-acid-long precursor to the subunit of β-amylase. An amino acid sequence identical to the N-terminal amino acid sequence of the mature subunit appeared immediate-ly after the initiator methionine of the precursor, indicating that the subunit of β-amylase is synthesized as a mature form. Comparison of the amino acid sequences of subunits of sweet potato β-amylase and seed β-amylases from barley and soybean indicated that these enzymes share about 68% amino acid identities among each other. Escherichia coli cells harboring the cDNA clone produced the mature-sized subunit of the, β-amylase, and the soluble extract exhibited β-amylolytic activity which migrated to the same position as the β-amylase purified from the sweet potato in non-denaturing polyacrylamide gel containing soluble starch indicating that oligomerization of the subunit occurred properly in E. coli cells.View full abstractDownload PDF (4836K) -
Etsuko Yasugi, Takeshi Kasama, Yousuke Seyama1991 Volume 110 Issue 2 Pages 202-206
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCeramide of the guinea pig Harderian gland was isolated and characterized. The purified ceramide gave two spots on thin-layer chromatography. Ceramide with the higher Rf value (NHCer) contained non-hydroxy fatty acids and that with the lower Rf value (HCer) contained 2-hydroxy fatty acids. The ratio of NHCer to HCer was 6:1. The non-hydroxy fatty acids of NHCer were composed of straight-chain acids (94.9%) and branched-chain acids (5.1%). The 2-hydroxy fatty acids were also composed of straight-chain acids (94.2%) and branched-chain acids (5.8%). The ratio of straight-chain acids to branched-chain acids was similar in NHCer and HCer. The long chain bases of NHCer and HCer consisted of straight chain sphinganines and sphingenines, and methyl-branched long chain bases. In NHCer, 59.9% of the total bases were methyl branched, and in HCer, 48.3%. The characteris-tics of ceramide, that is, the large amount of methyl-branched long chain bases and relatively small amount of methyl-branched fatty acids, are similar to those of cerebroside and sphingomyelin isolated from the same organ, although the ratios of constituents are different among these sphingolipids.View full abstractDownload PDF (2153K) -
Ching-Yuan Su, Henry Lardy1991 Volume 110 Issue 2 Pages 207-213
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFeeding the thermogenic steroid, 5-androsten-3β-o1-17-one (dehydroepiandrosterone, DHEA) in the diet of rats induced the synthesis of liver mitochondrial sn-glycerol 3-phosphate dehydrogenase to levels three to five times that of control rats within 7 days. The previously reported enhancement of liver cytosolic malic enzyme was confirmed. The induction of both enzymes was detectable at 0.01% DHEA in the diet, reached plateau stimulation at 0.1 to 0.2%, and was completely blocked by simultaneous treatment with actinomycin D. Feeding DHEA caused smaller, but statistically significant increases of liver cytosolic lactate, sn-glycerol 3-phosphate, and isocitrate (NADI+-linked) dehy-drogenases but not of malate or glucose 6-phosphate dehydrogenases. The capability of DHEA to enhance mitochondrial glycerophosphate dehydrogenase and malic enzyme was influenced by the thyroid status of the rats; was smallest in thyroidectomized rats and highest in rats treated with triiodothyronine. 5-Androsten-3β, 17β-diol and 5-androsten-3β-o1-7, 17-dione were as effective as DHEA in enhancing the liver mitochondrial glycero-phosphate dehydrogenase and malic enzyme. Administering compounds that induce the formation of cytochrome P450 enzymes enhanced liver malic enzyme activity but not that of mitochondrial glycerophosphate dehydrogenase. Arochlor 1254 and 3-methylcholanthrene also increased the response of malic enzyme to DHEA feeding.View full abstractDownload PDF (892K) -
Yasutada Imamura, Masao Kawakita1991 Volume 110 Issue 2 Pages 214-219
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInteraction of Ca2+ and Gd3+ ions with Ca2+-transporting ATPase of the sarcoplasmic reticulum (SR-ATPase) was analyzed. Binding of Ca2+ to the transport site caused an enhancement of intrinsic fluorescence of SR-ATPase. Gd3+ also induced fluorescence enhancement. However, the effects of Ca2+ and Gd3+ were additive rather than competitive, indicating that the Gd3+-binding site responsible for this enhancement is distinct from the Ca2+-transport site. Gd3+ ions at concentrations higher than 10 μM caused a marked fluorescence quenching, indicating an additional interaction at low-affinity binding sites. Interaction of Ca2+ with the transport site led to a quenching of fluorescence of N-(1- anilinonaphthy1-4) maleimide (ANM) covalently attached at SHN [as defined in Yasuoka-Yabe, K. & Kawakita, M. (1983) J. Biochem. 94, 665-675]. In this case the effects of Ca2+ and Gd3+ were mutually exclusive, indicating that Ca
and Gd3+ were competing for the same binding site (i.e. the transport site) to affect ANM fluorescence. Competition between Ca2+ and Gd3+ for the Ca2+-transport site was also demonstrated by direct measurement of Ca2+-binding using nitrocellulose membrane filters. Affinity of Gd3+ for the Ca2+-transport site was a little lower than that of Ca2+. Based on these results it was concluded that Gd3+ has at least three kinds of binding sites on SR-ATPase, namely the Ca2+-transport site, the Gd3+-specific high-affinity site, and a number of low-affinity sites. Gd3+ may be a useful Ca2+ analog to probe the Ca2+ -transport site of SR-ATPase, but one should be careful to avoid complications resulting from interactions at high-affinity as well as low-affinity Gd3+-binding sites which are distinct from the Ca2+-transport site.View full abstractDownload PDF (744K) -
Yasutada Imamura, Masao Kawakita1991 Volume 110 Issue 2 Pages 220-225
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGd3+ ions were bound to the Ca2+-transport site of Ca2+-transporting ATPase of the sarcoplasmic reticulum (SR-ATPase) and their effect on the ESR spectrum of spin-probes, which were attached to specific sites on SR-ATPase and embedded in the membranous lipid at various depths from the surface of the membrane, was studied. Spin-labeled reagents, 1-oxy1-2, 2-dimethyl-oxazolidine derivatives of maleimidoethyl-keto stearate, collectively abbreviated as MSL (m, n) were mainly used for labeling SR-ATPase. They have Cm-and Cn-hydrocarbon chains, respectively, on both sides of the spin label, of which the Cm-hydrocarbon chain is located distal to the carboxyl group of the keto stearate moiety. Paramagnetic interaction between Gd3+ and a spin probe was detected by measuring the decrease in the intensity of the ESR signal of the probe. Displacement of Gd3+ from the Ca2+-transport site by Ca2+, which had been confirmed previously by using fluorescently labeled SR-ATPase (described in the preceding article), led to a significant reversal of the paramagnetic effect of Gd3+ on MSL(12, 3) and MSL(10, 5) attached to SR-ATPase. On the other hand, the effect of Gd3+ was not reversed by Ca2+ when SR-ATPase labeled with MSL (1, 14) or a spin-label specific for the cytoplasmic domain was used. These results led us to conclude that the Ca2+-transport site of SR-ATPase is located in the membranous region of the molecule, but that the site is not very far from the surface of the membrane of the sarcoplasmic reticulum.View full abstractDownload PDF (775K) -
Da-Wei Gong, Shogo Ozawa, Yasushi Yamazoe, Ryuichi Kato1991 Volume 110 Issue 2 Pages 226-231
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrom liver cytosols of male Sprague-Dawley rats, two N-hydroxyarylamine sulfotransferases (HASTs) which sulfate N-hydroxy-2-acetylaminofluorene and a phenolsulfotransferase (PST-I) have been purified about 290-, 690-, and 210-fold, respectively, by the use of DEAE-anion exchange, Blue-Sepharose CL-6B, DEAE-HPLC, and ATP-agarose affinity chromatography. All three enzymes showed almost the same molecular weight of 33 kDa on SDS-polyacrylamide gel electrophoresis. In Western blots using antibody raised against HAST I, the two HASTs, but not PST-I, were detected. The content of total HAST in male rats was estimated as 4-5μg/mg cytosolic protein, about 4 times higher than that in female rats. Direct comparison of the DEAE-HPLC elution profiles of cytosol showed that HAST I and HAST II were male-dominant and male-specific, respectively, in their expression in the livers. Hypophysectomy decreased the level of HAST by 60% in male rats, but had no obvious effect in female rats. Intermittent injection of growth hormone to mimic the male secretory pattern raised the content in hypophysectomized rats of both sexes close to that in intact male rats, while the continuous infusion of growth hormone to mimic the female secretory pattern showed limited effects. In addition, the administration of triiodothyronine stimulated HAST in hypophysectomized rats of both sexes, and the extent of stimulation was nearly the same as observed in the male-type growth hormone treatment. PST-I, in contrast to HAST, showed no clear sex-related difference in hepatic content, and was not apparently affected by growth hormone or triiodothyronine. These results demonstrate that at least two forms of sulfotransferases are under the regulation of pituitary growth hormone and thyroid hormone. The experiment using hypophysectomized rats also indicates the principal role of growth hormone in the expression of the two HASTs in the male rat liver.View full abstractDownload PDF (3204K) -
Momoyo Kubota, Kazuhiro Sogawa, Yoshiko Kaizu, Takuji Sawaya, Junko Wa ...1991 Volume 110 Issue 2 Pages 232-236
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleotide sequence of the 5'-upstream region up to about-4.1 kb of the human P-450c gene was determined. Two kinds of repetitive sequences were located; one was the Alu sequence which was inserted at three positions (-3127 to -3038, -3017 to -2770, and -2167 to -1851), and the other was the SINE-R element located just upstream of the most distal Alu sequences. The region other than the two repeated sequences showed an overall similarity of 70% to that of the rat P-450c gene. Survey of XRE or its homologues, responsible for the inducible expression of the rat P-450c gene, revealed eight XRE core sequences in this region of the human P-450c gene. Three of them were carried in the Alu sequences. A fusion gene which was constructed by ligating the upstream region of the human P-450c gene to the chloramphenicol acetyltransferase (CAT) gene expressed the CAT activity in response to the inducer, methylcholanthrene, when transfected into Hepa-1 cells. Stepwise decrease in CAT activity in three regions was observed as the 5'-upstream sequence containing XRE motifs was removed. However, the XRE core sequence in the Alu sequences seemed inactive, because elimination of the three elements in the Alu sequences did not affect the expressed CAT activity. In accordance with this observation, competition experiments using gel mobility shift assay showed that XRE core sequences in the Alu sequences could not compete with the XRE sequence for the inducer-bound receptor. Other XRE motifs than those in the Alu sequences appeared active and were conserved at similar positions to their counterparts in the rat P-450c gene.View full abstractDownload PDF (1626K) -
Yasuo Watanabe, Masanao Shiramizu, Youichi Tamai1991 Volume 110 Issue 2 Pages 237-240
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA plasma membrane H+-ATPase gene from the salt-tolerant yeast Zygosaccharomyces rouxii was isolated by probing the genomic DNA library with a labeled DNA fragment derived from the Saccharomyces cerevisiae H+-ATPase gene (PMA1) and the nucleotide sequence of a cloned gene was determined. The gene encoded a polypeptide of molecular weight 100, 060 that consisted of 920 amino acid residues. The deduced amino acid sequence was 83% homologous to that of S. cerevisiae H+-ATPase and was highly similar to the conserved sequences in the plasma membrane ATPase family comprising yeast plasma membrane H+-ATPases. The peptide motifs involved in the ATPase functions were found in the Z. rouxii H+-ATPase sequence. The result of Northern analysis indicated that the size of the transcript of the Z. rouxii H+-ATPase gene was the same as that of S. cerevisiae PMA1.View full abstractDownload PDF (534K) -
Xianghong Zhu, Atsushi Hara, Tamotsu Taketomi1991 Volume 110 Issue 2 Pages 241-245
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman, porcine, goat, sheep, bovine, horse, canine, rat, mouse, guinea pig, and chicken serums were investigated for the existence of sulfatide. Among the ten mammal serums, seven were found to be sulfatide positive, and the amounts of sulfatide were determined to be: 16.29nmol/ml serum (porcine), 9.39 (bovine), 12.71 (goat), 7.75 (horse), 1.21 (sheep), 0.64 (human), and 0.16 (dog). The existence of sulfatide in the serums of human, goat, sheep, cow, horse, and dog is here reported for the first time. It is suggested that sulfatide is widely distributed in the serums of various mammals except for rodents and that it takes part in the anticoagulant systems. The fatty acids of those sulfatides comprised mainly non-hydroxy fatty acids and a significant amount (18-53% of the total fatty acid) of hydroxy fatty acids with chain lengths of C16, C22, C23, and C24. The long chain bases comprised sphingenine, sphinganine, and 4-D-hydroxysphinganine. Experiments to elucidate the mechanism of the anticoagulant activity of sulfatide revealed that it was specific to sulfatide and that the galactose-bound sulfate group is essential for this activity. The activity of clusters of sulfatide molecules was much more pronounced than that of single molecules.View full abstractDownload PDF (2005K) -
Akiho Yokota1991 Volume 110 Issue 2 Pages 246-252
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cause of the inflection in the course of the carboxylase reaction and the changes in the functioning form of spinach ribulose bisphosphate carboxylase (RuBisCO) during the reaction were elucidated by relating the activity to the protein conformation of RuBisCO using a fluorescence probe, 2-p-toluidinylnaphthalene sulfonate. The activity of RuBisCO in the linear phase was 50 to 60% of that in the initial burst at 0.5 to 1.0 mM ribulose bisphosphate (RuBP) and 65 to 80% at 2 to 5 mM RuBP. The amount and the progress of the decrease in the activity during the reaction had a close relationship to a change in the protein conformation of RuBisCO. The enzyme, the substrate binding sites of which were masked beforehand with carboxyarabinitol bisphosphate, still showed a change of its protein conformation upon addition of RuBP, suggesting that RuBisCO has two (substrate and regulatory) RuBP-binding sites per RuBisCO protomer. RuBisCO required over 2 mM RuBP for binding on the regulatory sites. Both sites also bound 6-phosphogluconate. When both sites were masked with 6-phosphogluconate beforehand, the course of the subsequent carboxylase reaction was linear with time. From these results, I propose that the inflection in the course of the reaction of spinach RuBisCO is a hysteretic response of the enzyme to RuBP bound to both substrate and regulatory sites.View full abstractDownload PDF (909K) -
Akiho Yokota, Masaya Higashioka, Akira Wadano1991 Volume 110 Issue 2 Pages 253-256
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRibulose bisphosphate carboxylase (RuBisCO) binds carboxyarabinitol bisphosphate (CABP) on its regulatory sites [Yokota, A. (1991) J. Biochem. 110, 246-252]. The characteristics of the equilibrium binding of CABP to the sites were examined by the gel-filtration method. Since RuBisCO binds CABP on the substrate sites with a dissociation constant of less than 10 pM, CABP bound exclusively to the substrate sites at less than 5 μM. Plotting the number of CABP bound to the sites other than the substrate sites against the concentration of CABP gave a typical “bumpy” curve; the binding number in the intermediate plateau at 20 to 40 μM CABP was 3.7 to 4.4 mol per mol of RuBisCO and that at the saturating concentration of CABP was 7.6 to 7.8 mol per mol of RuBisCO. The Hill plot of their relationship gave a line which bent strongly at 20 to 40 μM CABP. The best fitting of the data to the equations derived from the binding model constructed according to the reported model [Teipel, J. & Koshland, D. E., Jr. (1969) Biochemistry 8, 4656-4663] showed that the binding of CABP to the regulatory sites proceeded with positive cooperativity both before and after the plateau. The dissociation constant decreased from 31 to 14 μM by the factor of 1/1.3 in the former group and 490 to 0.7 μM by the factor of 1/8.9 in the latter with increasing binding number of CABP.View full abstractDownload PDF (465K) -
Katsumi Ohyama, Ichiro Tokimitsu, Shingo Tajima1991 Volume 110 Issue 2 Pages 257-260
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo study how collagen synthesis is regulated in developing chick embryonic skin, hydroxyproline synthesis, incorporation of proline, and translational activity and content of collagen mRNA in 12-, 15-, and 18-day skins were determined and compared with each other. Hydroxyproline synthesis in the 18-day skins was markedly increased over that in the 12-day skins, whereas proline incorporation was moderately increased. The increase in collagen synthesis from day 15 to 18 was accompanied by increases in both the translational activity and the content of type I procollagen mRNA, with a selective increase in the lower-molecular-weight species of proα1 (I) collagen mRNA. In contrast, the stimulation of collagen synthesis from day 12 to day 15 did not parallel the levels of type I procollagen mRNA. These results suggest that the stimulation of collagen synthesis is regulated by collagen mRNA levels only in the later stage of development (from day 15 to day 18). Both the collagen synthesis and type I procollagen mRNA levels in the fibroblasts isolated on each corresponding day were constant. The difference in collagen synthesis under two different culture conditions suggests that cell-matrix interaction and/or some serum factors, including several growth factors, are essential for the marked stimulation of collagen synthesis observed in 12- to 18-day skin.View full abstractDownload PDF (2936K) -
Takayuki Hara, Toshiyuki Miyata1991 Volume 110 Issue 2 Pages 261-266
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cross-linked complex between bovine NADPH-adrenodoxin reductase (AR) and adrenodoxin (AD) was prepared with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and purified, as described previously [Hara, T. & Kimura, T. (1989) J. Biochem. 105, 594-600]. The covalent complex was S-pyridylethylated and digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase HPLC to identify the cross-linked peptide. Comparison of the HPLC chromatograms of the peptides showed that (i) two tandem peptides (K-4 and K-5) from AD and a peptide (K-1) from AR were missing in the chromatogram of the peptides of the covalent complex and (ii) a single new peak was observed in the chromatogram of the peptides from the covalent complex. Amino acid composition and sequence analyses showed that the newly observed peptide was a covalently cross-linked peptide formed between a peptide K-4-K-5 (Ile-25-Lys-98) derived from AD and a peptide K-1 (Ser-l-Lys-27) derived from AR, in which an amide bond had been formed between the ε -amino group of Lys-66 in AD and the γ-carboxyl group of Glu-4 in AR. These results indicate that the binding site of AR with AD is localized in the amino-terminal part of AR and that of AD with AR is localized around Lys-66 of AD. The six clustered basic amino acid residues (His-24, Lys-27, His-28, His-29, Arg-31, and His-33) present in the amino-terminal portion of AR and the eight clustered acidic amino acid residues (Glu-65, Glu-68, Asp-72, Glu-73, Glu-74, Asp-76, Asp-79, and Asp-86) present in the middle part of AD may play an important role in the complex formation.View full abstractDownload PDF (745K) -
Isao Yumoto, Yoshihiro Fukumori, Tateo Yamanaka1991 Volume 110 Issue 2 Pages 267-273
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe membrane fraction of the facultative alkalophilic bacterium, Bacillus YN-2000, was found to contain considerably larger amounts of two c-type cytochromes, cytochromes c-553 and c-552, when the bacterium was grown at pH 10 than when it was grown at lower pHs (pH 7-9). In particular, cytochrome c-553 was present in a much higher amount in the cells grown at pH 10 than in those grown at pH 8. Cytochromes c-553 and c-552, which are membrane-bound proteins, were purified to electrophoretically homogeneous states from Bacillus YN-2000. Cytochrome c-553 showed absorption peaks at 553, 524, and 417nm in the reduced form, and a peak at 411nm in the oxidized form. Its molecular weight was estimated to be 10, 500 from the results of SDS-polyacrylamide gel electrophoresis. However, its molecular weight was estimated to be 127, 000 by gel filtration. Therefore, it seemed to occur as an oligomer in solution. The isoelectric point of cytochrome c-553 was determined to be 3.9. Its midpoint redox potential was found to be + 87 mV in the pH region from 6 to 8. Cytochrome c-553 reacted with cytochrome c oxidase of the bacterium and the reaction was greatly accelerated in the presence of poly-L-lysine. Cytochrome c-552 showed absorption peaks at 552, 521, and 416nm in the reduced form, and a peak at 408nm in the oxidized form. The cytochrome molecule seemed to be composed of six different subunits, with molecular masses of 40, 32, 19, 17, 14, and 12kDa, respectively. Heine bound to the 14 and 12kDa subunits, respectively. The molecular weight of cytochrome c-552 was estimated to be 143, 000 by gel filtration. On the basis of these results, it seemed to be composed of one molecule each of the six polypeptides. Its isoelectric point was found to be 4. 0 and its midpoint redox potential at pH 7 to be +91mV. Cytochrome c-552 did not react with cytochrome c oxidase of the bacterium.View full abstractDownload PDF (2096K) -
Keiko Nohara, Minoru Suzuki, Fuyuhiko Inagaki, Kunimitsu Kaya1991 Volume 110 Issue 2 Pages 274-278
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe thin-layer chromatographic (TLC) pattern of gangliosides of rat thymocytes showed a profile characterized by the occurrence of a predominant ganglioside which did not correspond to any reference gangliosides of rat brain. The ganglioside was isolated from rat thymus, and characterized by compositional analysis, methylation analysis, sialidase treatment, negative-ion fast atom bombardment (FAB) mass spectrometry, and proton NMR spectroscopy. The structure was elucidated to be NeuGcα2-8NeuGcα2-3Galβ1- 3GalNAcβ1-4Galβ1-4G1cβ1-1Cer. This is the major ganglioside of rat thymus lymphoid cells and is one of the GM1b-derived gangliosides, GD1c, having two N-glycolylneuraminic acids. This is the first report on the occurrence of GD1c in normal animal cells.View full abstractDownload PDF (3084K) -
Hirohide Toyama, Nobuyoshi Esaki, Tohru Yoshimura, Katsuyuki Tanizawa, ...1991 Volume 110 Issue 2 Pages 279-283
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe guanidine hydrochloride-induced subunit dissociation and unfolding of thermostable alanine racemase from Bacillus stearothermophilus have been studied by circular dichroism, fluorescence and absorption spectroscopies, and gel filtration. The overall process was found to be reversible: more than 75% of the original activity was recovered upon reduction of the denaturant concentration. In the range of 0.6 to 1.5M guanidine hydrochloride, the dimeric enzyme was dissociated into a monomeric form, which was catalytically inactive. The monomeric enzyme appeared to bind the cofactor pyridoxal phosphate by a non-covalent linkage, although the native dimeric enzyme binds the cofactor through an aldimine Schiff base linkage. The monomer was mostly unfolded, with the transition occurring in the range of 1.8 to 2.2M guanidine hydrochloride.View full abstractDownload PDF (510K) -
Fusao Kimizuka, Yuki Taguchi, Yoichi Ohdate, Yasutoshi Kawase, Tomoko ...1991 Volume 110 Issue 2 Pages 284-291
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.View full abstractDownload PDF (4242K) -
Taishin Takuma, Tokuro Ichida1991 Volume 110 Issue 2 Pages 292-294
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRp-cAMPS, the Rp-diastereomer of adenosine 3', 5'-phosphorothioate, is often referred to as a cAMP antagonist, since it binds to the regulatory subunit of cAMP-dependent protein kinase without dissociation of free catalytic subunits. To evaluate the role of cAMP-dependent protein kinase in amylase exocytosis, we examined the effect of Rp-cAMPS on amylase release from rat parotid acini. Rp-cAMPS did not stimulate amylase release from saponin-permeabilized parotid acini, whereas its Sp-isomer strongly evoked amylase release. Rp-cAMPS dose-dependently inhibited amylase release stimulated by Sp-cAMPS. In the presence of Rp-cAMPS, the dose-response curve of Sp-cAMPS was shifted to the right. The inhibitory effect of Rp-cAMPS on isoproterenol-induced amylase release was not detected in intact acini, but was clearly observed in the permeabilized ones. Rp-cAMPS markedly inhibited protein phosphorylation evoked by Sp-cAMPS, indicating that Rp-cAMPS prevents the dissociation of cAMP-dependent protein kinase. These results, taken topther with synergistic increase in amylase release by the combination of site-selective cAMP analogues [T. Takuma (1990) J. Biochem. 108, 99-102], suggest that cAMP-dependent protein kinase is involved in the exocytosis of amylase from parotid acini.View full abstractDownload PDF (1242K) -
Takashi Nakazawa, Fumio Sakiyama1991 Volume 110 Issue 2 Pages 295-300
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe indole C-2(δ1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with l3C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed dehydration. [δ1-13C]Trp 62-lysozyme in which Trp 62 is labeled with 90% 13C has the same chemical and enzymatic properties as the native protein. The reverted lysozyme gave a single 13C-NMR signal at 125 ppm. pH-titration of the 13C signal indicated a transition at pH 3.9 for the free enzyme. In the presence of (GleNAc)3, the resonance signals were shifted 0.5-1 ppm upfield, and the transitions in the titration curve were observed at pH 3.9 and 6.5. Asp 52 and Glu 35 were assigned to the groups with pKas of 3.9 and 6.5, respectively. In [2-13C] AHT 62-lysozyme, which has 3-(2-amino-3-hydroxy-3H- [2-13C]indo1-3-yl)alanine (AHT) at position 62, AHT 62 behaved quite differ-ently from Trp 62 on pH-titration of the 13C-label. These results suggest that a confor-mational change around Trp 62 is induced upon ionization of the catalytic residue and that the structural flexibility of the side chain of this aromatic residue in the substrate binding site is closely related to the function of lysozyme.View full abstractDownload PDF (698K) -
Masaya Ito, Yukari Hirono, Satoshi Watanabe, Hisashi Yamamoto, Koscak ...1991 Volume 110 Issue 2 Pages 301-305
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSImmunofluorescence microscopy showed that a monoclonal antibody, 2F3, specific to the βI subunit (Cap Zα) of β-actinin (Cap Z) bound to the Z lines of chicken breast muscle. When myofibrils were briefly extracted with 0.6 M KI, the reactivity of the Z lines with 2F3 was very weak, but on subsequent treatment with purified β-actinin, the antibody binding recovered. β-Actinin inhibited elongation of the actin filaments of isolated I-Z-I brushes, myosin-extracted sarcomeres, on the addition of G-actin. However, when an increased concentration of G-actin was added, the inhibitory action of β-actinin became negligible, suggesting that β-actinin did not cap the pointed end of an actin filament in a myofibril.View full abstractDownload PDF (4905K) -
Hiroyuki Tachikawa, Tadashi Miura, Yoshio Katakura, Takemitsu Mizunaga1991 Volume 110 Issue 2 Pages 306-313
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDIl gene is essential for cell growth.View full abstractDownload PDF (4215K)
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